Cholangiopathies are incurable biliary tree disorders characterized by tissue injury with neutrophil recruitment, cholangiocyte hyperproliferation, and stromal cell expansion. Signals driving extrahepatic bile duct (EHBD) inflammation and proliferation are poorly understood. This application aims to answer fundamental questions about injury-driven processes promoting biliary proliferation. The rationale for these studies is that understanding molecular mechanisms of EHBD inflammatory and proliferative responses can inform therapeutic strategies to prevent and treat complications of cholangiopathies. My preliminary data shows a massive increase in cholangiocyte proliferation within 24 hours after acute injury. Further, increased cholangiocyte Hedgehog (HH) ligand targets stromal cell CXCL1, which recruits neutrophils to the injured tissue. My project focuses on uncovering the mechanisms of this complex crosstalk involving epithelial cholangiocyte – stromal fibroblast – neutrophil signaling to promote biliary proliferation and facilitate tissue repair. Biliary hyperproliferation is associated with HH-dependent expression of FOSL1, a transcription factor known to mediate proliferative responses in other tissues. IL1β is increased post EHBD injury in neutrophils and can regulate FOSL1. The overarching hypothesis for this proposal is that in response to EHBD injury, cholangiocytes secrete Indian HH which induces GLI1+ fibroblasts to produce CXCL1, leading to neutrophil recruitment and promotion of cholangiocyte proliferation through a IL1β/FOSL1-dependent mechanism. I will use state-of-the art in vivo genetic mouse and pharmacologic approaches combined with ex vivo human and mouse organoid and primary culture models to define the molecular mechanisms of cellular crosstalk regulating EHBD repair. Bile duct ligation (BDL) will be used as an acute biliary injury model. CUT&RUN chromatin mapping, cell isolation by flow cytometry, and RNA-seq approaches will be conducted within this proposal. Findings will be validated using archived biliary tissues from patients with/without cholangiopathy. Aim 1 will focus on GLI1+ stromal cells and regulation of Cxcl1 expression to determine if HH signaling induces fibroblasts to express Cxcl1 via direct transcriptional activation by GLI1. This aim will use reporter mice to isolate GLI1+ cells for downstream in vivo and ex vivo analyses. Pharmacological activators and inhibitors of HH signaling will be used to test effects of GLI1 modulation on gene expression. Aim 2 will focus on neutrophil-derived pro-proliferative signals to test whether neutrophils promote biliary proliferation via a IL1β/FOSL1-dependent mechanism and identify candidate neutrophil-derived factors. Aim 2 will use reporter mice to isolate neutrophils for downstream in vivo and ex vivo analyses. A genetic mouse model will test effects of Fosl1 deletion on biliary proliferation post EHBD injury. This proposal will allow me to gain proficiency in state-o...